The present invention relates to a pretreatment method of a sample for detecting HBs antigen, which is a surface antigen of the hepatitis B virus, and a method for detecting HBs antigen utilizing the pretreatment method. The present invention also relates to a pretreatment reagent kit for detecting HBs antigen.
Hepatitis B is a liver disease caused by the hepatitis B virus (HBV). HBV is a spherical virus which is mainly composed of an envelope portion and a core portion. The envelope portion contains HBs antigen and the core portion contains genomic DNA, HBc antigen, HBe antigen and the like. After infection with HBV, HBs antigen is released into the circulation of the host and the amount thereof increases with development of hepatitis. Therefore in the test for hepatitis B, blood HBs antigen is first measured. A positive test result for HBs antigen indicates that the subject is infected with HBV. In contrast, the result of HBs antigen negative indicates that the subject is not infected with HBV. Meanwhile after onset of hepatitis B, HBs antibody is started to be produced in vivo in the host, that is a neutralizing antibody of HBs antigen. In the test for hepatitis B, infection history of the subject can be examined by simultaneously measuring HBs antibody. It is considered clinically that hepatitis B is cured when a subject is HBs antigen negative and HBs antibody positive.
However, recent studies have revealed that even after remission of hepatitis B, some HBV genes remain in liver and peripheral mononuclear cells. It has also recently been shown that application of an immunosuppressant to patients with malignant tumor or rheumatoid patients who have HBV infection history causes HBV reactivation and thus severe hepatitis. This type of hepatitis is referred to as “de novo hepatitis B” and is found to be highly frequently fulminated and have high mortality compared to normal hepatitis B.
It is considered that in order to prevent de novo hepatitis B from being fulminated, initiation of treatment at an early stage of the onset is effective. Therefor it is important to detect reactivation of the virus at the early stage. However, it is considered that at the early stage of reactivation the amount of the HBV virus is still minute and HBs antibody from a patient also exists. As HBs antibody from patients binds to HBs antigen to inhibit the detection of the antigen, a test having extremely high sensitivity is required for early detection of reactivation of HBV.
A known test of HBV with high detection sensitivity utilizes PCR. In the test, HBV DNA is amplified to detect the HBV. However, the test is generally and mostly carried out at institutions that can carry out the test after sending samples to the institutions, and thus it takes long time to have the test result available. In addition, the test is expensive.
Meanwhile a HBs antigen test by immunological means requires a short time to have the test result available compared to the test by PCR and is inexpensive. A method for detecting HBs antigen by immunological means is disclosed in WO 2006/033368, for example, in which a sample is pretreated with a denaturing agent such as an alkaline agent or a surfactant to denature HBs antibody from a patient followed by detection of HBs antigen using a certain anti-HBs antibody for detection.